Tetraazamacrocycles such as derivatives of cyclam (1,4,8,11-tetraazacyclotetradecane) generate an important interest in many fields such as medicine, especially nuclear medicine; epuration of effluents contaminated with radioactive elements or metals such as lead; catalysis; solid/liquid extraction and liquid/liquid extraction; or detection of traces of metallic cations. The present invention relates to all these fields of applications, especially nuclear medicine.
In nuclear medicine, radiopharmaceuticals used as therapeutic agents or as imaging agents often comprise chelates of radioelements. To improve the efficiency of radiopharmaceuticals, a targeting biomolecule may be appended on the chelating moiety in order to induce a site-specific delivery of the radiation, leading to a bifunctional chelating agent (BCA). Obtaining a BCA requires the introduction of an appropriate conjugation group in the structure of the metal chelator, to allow for the bioconjugation prior or after labeling with the radioisotope. The targeting agent may be for example an antibody, an hapten or a peptide. Depending on the nature of the radionuclide, it is for example possible to perform PET imaging (Positron Emission Tomography), SPECT (Single Photon Emission Computed Tomography) or RIT (RadiolmmunoTherapy).
For applications in nuclear medicine, the chelate should thus be bioconjugated to a biological vector while trapping the radionuclide to form a stable complex preventing the release of the metal in the organism. Moreover, when using radioactive emitters, the kinetic constraint has to be considered because of the limited half-life of the radionuclide.
Dota (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) is a tetra N-functionalized cyclen (scheme 1). In scheme 1, dota is referred to as “H4dota”, the four hydrogen atoms specified before “dota” reflecting the fact that in order to have the four carboxylic acid functions in “COOH” form, the four amines of the macrocycle should be protonated. The same nomenclature is used along the description for macrocycles comprising carboxylic acid functions.
Dota is the most used ligand to complex gadolinium (III) for MRI imaging. Dota also enables to complex other metals commonly used in nuclear medicine, such as for example 111In, 68Ga, 149Tb, 213Bi, 212Bi, 212Pb, 64Cu or 67Cu. Derivatives of the dota, are today widely studied (scheme 1).

Among the range of potentially useful metals in nuclear medicine, copper has been receiving much interest due to the existence of several radionuclides with different half-life times and emission properties suitable for diagnostic imaging or therapeutic applications. The most interesting nuclides are 67Cu (t1/2=62.0 h, β− 100%, Emax=0.577 MeV) for radiotherapy, and 64Cu (t1/2=12.7 h, β+17.4%, Emax=0.656 MeV, β− 39.6%, Emax=0.573 MeV) for both positron emission tomography (PET) and radiotherapy. Copper exists predominantly as divalent metal cation that prefers donor groups such as amines and anionic carboxylates to form complexes with coordination numbers of 4-6. High coordination numbers are usually preferred, often providing square pyramidal, trigonal bipyramidal or octahedral geometries, so as to entirely surround the metal cation. Within the vast range of acyclic and cyclic ligands successfully used for copper complexation, the family of tetraaza macrocycles with N-appended coordinating arms stands out owing to the efficiency and versatility of its copper chelation.
Like copper, gallium prefers high coordination numbers, especially under the form of octahedral geometries and tetraaza macrocycles with N-appended coordinating arms may be used for its chelation. The most interesting nuclide for nuclear imaging is 68Ga (t1/2=68 min, β+100%, Emax=2.921 MeV), for positron emission tomography (PET).
The following requirements are commonly admitted in the art as specifications for an optimized chelate intended to be used in nuclear medicine:                a) rapid metallation kinetics with respect to the time of the radionuclide half-life, even under the acidic conditions in which most radionuclides are produced;        b) a very good thermodynamic stability;        c) inertness with respect to other metals, especially Zn2+ which is present in high amounts in the biological medium or as byproduct of radionuclides production such as 64Cu;        d) kinetic inertness;        e) stability upon reduction in the biological media of the chelated metal, such as for example the stability of copper (I) complex as a reduced form of the initially chelated copper (II).        
Metallation kinetics (point a) may be determined using UV-visible spectrometry by measuring the increasing intensity of the complex d-d transition band. When possible, i.e. depending on whether the metal is paramagnetic or not, metallation kinetics may also be determined by NMR. Suitable metallation kinetics depends on the half-life of the radionuclide used to form the chelate.
Thermodynamic stability (point b) may be evaluated by determining the stability constants of the complexes, especially the association constant K and pK (or log K).
Stability constants may be measured by potentiometry or spectroscopies. pM values may be calculated from pK in order to compare thermodynamic stability with corresponding values of other ligands of the prior art. Indeed, pM reflects the amount of ligand not chelated, taking into account the basicity of the ligand. In the present invention, a “very good thermodynamic stability” refers to a thermodynamic stability at least comparable, preferably better than that of the dota chelate formed with the same metal.
Inertness with respect to other metals (point c) may be evaluated by determining and comparing the pCu2+ versus pZn2+. Competitive experiments may also be conducted. Especially, excess of zinc necessary to lead to a transchelation may be determined in competitive experiments with zinc. In the present invention, a chelate is considered having a suitable inertness with respect to other metals when it has inertness at least comparable, preferably better than that of the dota chelate formed with the same metal.
Kinetic inertness (point d) may be evaluated by measuring metal dissociation upon competition with H+, in acid medium. Especially, half-life of the complex may be determined in presence of H+ at different concentrations and temperatures. In the present invention, a chelate is considered having a suitable kinetic inertness when it is at least comparable, preferably better than that of the dota chelate formed with the same metal.
Stability upon reduction (point e) may be evaluated by determining the dissociation of the reduced metal. Dissociation may be measured with cyclic voltammetry in electrochemical experiments. In the present invention, a chelate is considered having a suitable stability upon reduction when it is at least comparable, preferably better than that of the dota chelate formed with the same metal.
Chelates with a good thermodynamically stability and a kinetic inertness prevent possible transchelation of the metal when the complex is challenged with biological ligands or bioreductants.
It is also important that the chelate and the chelator display good water solubility.
As stated above, the commercially available dota is used to complex 64Cu(II), 67Cu(II) and 68Ga(III). However, copper-dota chelates are far from meeting requirements of the above specifications.
Due to their good affinity with copper (II), tetraazacycloalkanes derivatives of cyclam, such as for example teta and te2a (scheme 1), were recently used to complex 64Cu or 67Cu for PET or RIT applications. Their suitable N-functionalization can also give them a good affinity toward other metals such as heavy metal or lanthanides and extend their use in these applications with for example 99mTc, 186Re, 188Re, 111In, 68Ga, 89Zr, 177Lu, 149Tb, 153Sm, 212B (212Pb), 213Bi and 225Ac. However, chelates formed from these derivatives of cyclam do not meet all requirements of the above specifications.
Therefore, there is a need for new ligands enabling to form chelates meeting all the requirements of the specifications mentioned above. Especially, ligands potentially useful for radiopharmaceuticals should combine a high thermodynamic stability and kinetic inertness of the complexes with a fast metal complexation under mild conditions, as the latter is crucial to take full advantage of the short radioisotope half-life times and allow for use of heat- and pH-sensitive biomolecules.
Picolinate arms have been demonstrated to induce strong coordination ability toward transition and post-transition metals when appended on macrocyclic ligands, as well as non macrocyclic ligands. Indeed, picolinate moiety is bidentate: it has a nitrogen atom and an oxygen atom, both capable to participate to the coordination of a metal. Therefore, picolinate derivatives were recently used for the complexation of lanthanides, lead or bismuth (Rodrigez-Rodrigez A. et al. Inorg. Chem. 2012, 51, 13419-13429; Rodrigez-Rodrigez A. et al. Inorg. Chem. 2012, 51, 2509-2521). They were also recently used for the complexation of copper.
Orvig et coll. disclosed a derivative of ethylenediamine grafted with two picolinate arms H2dedpa, represented on scheme 2 below for the chelation of copper (Boros et al., JACS, 2010, 132, 15726-33; Boros et al. Nucl. Med. Biol. 2011, 38, 1165-1174).

Derivatives of H2dedpa were also proposed, with various bioconjugation groups (Boros et al. Inorg. Chem. 2012, 51, 6279-6284; Bailey et al. Inorg. Chem. 2012, 51, 12575-12589; Boros et al. Nucl. Med. Biol. 2012, 39, 785-794). However, results were quite disappointing, especially for the coordination of Cu(II), for an application in medicine. Indeed, Cu(II) complexes display low kinetic and thermodynamic stability, as well as decreased serum stability (Boros et al. Inorg. Chem. 2012, 51, 6279-6284), thus not meeting requirements b), d) and e) of the above specifications.
In a preliminary work, the Applicant proposed a triaza macrocycle with one picolinate and two picolyl pendant arms, Hno1pa2py (scheme 3), which was found to easily form stable and inert copper(II) complexes as well, and additionally resulted in a very efficient radiolabeling with 64Cu (Roger et al. Inorg. Chem. 2013, 21(9), 5246-5259). Despite promising properties, all the requirements of the above-mentioned specifications were not entirely met: the stability of the formed copper chelate with this ligand needs to be improved, in particular upon the reduction of copper (II) to copper (I) in the physiologic media.

The Applicant then developed picolinate derivatives of cyclen and cyclam (scheme 3), especially a first generation of monopicolinate derivative of cyclam, Hte1pa (Lima et al. Inorg. Chem. 2012, 51(12), 6916-6927). The corresponding copper chelate gives good results relative to the requirements a)-c) of the specifications. However, inertness in acidic medium, (point d) of the specifications, and inertness with regard to reduction (point e) were not optimized.
Therefore, the Applicant conducted research to provide a new ligand comprising picolinate arms, overcoming abovementioned drawbacks, i.e. to improve inertness in acidic medium and inertness in reductive medium, while meeting the other requirements of the specifications mentioned above.
Rigid tetraazamacrocycles, known as “cross-bridged chelators”, are the subject of great interest due to the outstanding behavior of their complexes, especially their inertness.
Examples of cross-bridged chelators are cross-bridged cyclam derivatives cb-te2a and side-bridged sb-te1a1p or cross-bridged cyclen derivative cb-do2a (scheme 1). Cross-bridged chelators are defined as containing an ethylene (or propylene) bridging unit connecting two nitrogen atoms of the macrocycle in trans position and they have originated some of the most inert copper (II) complexes ever reported. Furthermore, successful radiolabeling and bioconjugation of a few examples have also been achieved.
Especially, cross-bridged cb-te2a attracts a great interest since it forms the most inert copper complexes (points d) and e) of above specifications), leading thus to limited if any release of copper in the body.
Therefore, the Applicant considered introducing a cross-bridge in Hte1pa, to form the new ligand Hcb-te1pa, in order to improve inertness:

However, all constrained bridged chelators described in the art, including Hcb-te2a, are very basic since they are proton-sponges: a proton remains blocked in the macrocyclic cavity due to the structure of the compound, and this proton may not be easily displaced by the metal. This proton-sponge behavior renders metallation kinetics very slow. Drastic conditions are necessary to displace the proton, such as elevated temperatures, which is incompatible when sensible biological vectors are grafted to the chelate to form a bioconjugate.
As a consequence, cross-bridged chelators, and especially Hcb-te2a, meet the above mentioned specifications, especially inertness points d) and e), with the notable exception of a very slow metallation kinetics (point a).
Therefore, by introducing a cross-bridge in Hte1pa to improve inertness, the Applicant expected a drastic decrease of metallation kinetics, leading to a ligand offering a compromise between good inertness and fast kinetics but not meeting all 5 requirements of the above specifications.
As expected, the Applicant demonstrated that, as other cross-bridged derivatives, the Hcb-te1pa ligand of the invention is a proton-sponge (see acido-basic studies—example 5, paragraph B.1).
However and unexpectedly, cross-bridging Hte1pa to form Hcb-te1pa and derivatives thereof did not lead to a decrease of metallation kinetic, compared to non-cross-bridged cyclams. On the contrary, the cross-bridged ligand of the invention Hcb-te1pa surprisingly shows a very rapid metallation kinetic, even in acidic conditions. The metallation occurs quasi instantaneously: for example, more than 90% copper is chelated immediately and remaining copper is chelated within a few seconds (see experimental part—example 5, paragraph B.3). To the knowledge of the Applicant, there is no other case reported in the art of a cross-bridged cyclam or cyclen having a rapid metallation kinetic in aqueous acidic medium and the present invention overcomes a strong prejudice of the skilled artisan.
Without willing to be linked by a theory, it seems that the pre-organized character of the cross-bridged ligand, which was evidenced in crystallographic studies (FIG. 1), together with the presence of a carbonyl group on the aromatic moiety, might be at the origin of this unexpected behavior. It was observed that the structure of the chelate is close to the structure of the ligand (FIG. 2).
The Applicant thus provides a new ligand of formula Hcb-te1pa:
and derivatives thereof, especially derivatives functionalized with coupling functions suitable for grafting vectoring groups or derivatives comprising vectoring groups.
In a preferred embodiment, the invention relates to ligands of formula (I)
                wherein n, R1, R2, R3, R4, R5, L1, L2, L3 and L4 are as defined below.        
Upon complexation with metallic cation, the ligands of the invention lead to chelates meeting the 5 requirements of the above specifications. Especially, properties of copper(II) chelate of Hcb-te1pa are reported in the experimental part below and compared to those of copper(II) chelates of the art, evidencing that the chelate of the invention entirely fulfills specifications.
The invention also relates to chelates resulting from the complexation of a ligand of formula (I) with metallic cations.
The ligand of formula (I) of the invention presents the advantage of being easily manufactured using a simple chemical synthesis.
Moreover, the ligand Hcb-te1pa and derivatives thereof present a competency for diverse radioisotopes useful in nuclear medicine, such as for example 64Cu, 67Cu, 68Ga, 89Zr, 99mTc, 111In, 186Re, 188Re, 210At, 212Bi (212Pb), 213Bi, 225Ac, 90Y, 177Lb, 153Sm, 149Tb or 166Ho.
The structure of Hcb-te1pa enables the bio-vectorization of the chelate by the introduction of vectorizing groups on the cyclam core, through N-functionalization and/or C-functionalization. Especially, the cyclam core may be C-functionalized according to the method described in patent application WO2013/072491. Moreover, the carboxylic function of the picolinate arm may also be functionalized. The invention thus encompasses Hcb-te1pa ligand, functionalized and/or vectorized derivatives thereof and corresponding chelates with metallic cations, preferably copper(II) or gallium(III).
The chelate of the invention is obtained in aqueous medium, contrary to what is currently done in the art, which is very advantageous for nuclear medicine applications.
Besides applications in nuclear medicine, the ligand of formula (I) of the invention may be used for epuration of effluents contaminated with radioactive elements or metals such as lead; catalysis; solid/liquid extraction and liquid/liquid extraction; or detection of traces of metallic cations.